Immune regulatory oligonucleotide (IRO) compounds to modulate toll-like receptor based immune response

ABSTRACT

The invention provides novel immune regulatory oligonucleotides (IRO) as antagonist of TLRs and methods of use thereof. These IROs have unique sequences that inhibit or suppress TLR-mediated signaling in response to a TLR ligand or TLR agonist. The methods may have use in the prevention and treatment of cancer, an autoimmune disorder, airway inflammation, inflammatory disorders, infectious disease, skin disorders, allergy, asthma or a disease caused by a pathogen.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.11/549,048, filed on Oct. 12, 2006, the contents of which areincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention generally relates to the field of immunology andimmunotherapy, and more specifically to immune regulatoryoligonucleotide (IRO) compositions and their use for inhibition and/orsuppression of Toll-like Receptor-mediated immune responses.

2. Summary of the Related Art

Toll-like receptors (TLRs) are present on many cells of the immunesystem and have been shown to be involved in the innate immune response(Hornung, V. et a.l, (2002) J. Immunol. 168:4531-4537). In vertebrates,this family consists of ten proteins called TLR1 to TLR10, which areknown to recognize pathogen associated molecular patterns from bacteria,fungi, parasites, and viruses (Poltorak, a. et al. (1998) Science282:2085-2088; Underhill, D. M., et al. (1999) Nature 401:811-815;Hayashi, F. et. al (2001) Nature 410:1099-1103; Zhang, D. et al. (2004)Science 303:1522-1526; Meier, A. et al. (2003) Cell. Microbiol.5:561-570; Campos, M. A. et al. (2001) J. Immunol. 167: 416-423; Hoebe,K. et al. (2003) Nature 424: 743-748; Lund, J. (2003) J. Exp. Med.198:513-520; Heil, F. et al. (2004) Science 303:1526-1529; Diebold, S.S., et al. (2004) Science 303:1529-1531; Hornung, V. et al. (2004) J.Immunol. 173:5935-5943). TLRs are a key means by which mammals recognizeand mount an immune response to foreign molecules and also provide ameans by which the innate and adaptive immune responses are linked(Akira, S. et al. (2001) Nature Immunol. 2:675-680; Medzhitov, R. (2001)Nature Rev. Immunol. 1:135-145). TLRs have also been shown to play arole in the pathogenesis of many diseases, including autoimmunity,infectious disease, and inflammation (Cook, D. N. et al. (2004) NatureImmunol. 5:975-979) and the regulation of TLR-mediated activation usingappropriate agents may provide a means for disease intervention.

Some TLRs are located on the cell surface to detect and initiate aresponse to extracellular pathogens and other TLRs are located insidethe cell to detect and initiate a response to intracellular pathogens.Table 1 provides a representation of TLRs and the known agoniststherefore (Diebold, S. S. et al. (2004) Science 303:1529-1531; Liew, F.et al. (2005) Nature 5:446-458; Hemmi H et al. (2002) Nat Immunol3:196-200; Jurk M et al., (2002) Nat Immunol 3:499; Lee J et al. (2003)Proc. Natl. Acad. Sci. USA 100:6646-6651); (Alexopoulou, L. (2001)Nature 413:732-738).

TABLE 1 TLR Molecule Agonist Cell Surface TLRs: TLR2 bacteriallipopeptides TLR4 gram negative bacteria TLR5 motile bacteria TLR6 grampositive bacteria Endosomal TLRs: TLR3 double stranded RNA viruses TLR7single stranded RNA viruses TLR8 single stranded RNA viruses TLR9unmethylated DNA

Certain unmethylated CpG motifs present in bacterial and synthetic DNAhave been shown to activate the immune system and induce antitumoractivity. (Tokunaga T et al., J. Natl. Cancer Inst. (1984) 72:955-962;Shimada S, et al., Jpn. H cancer Res, 1986, 77, 808-816; Yamamoto S, etal., Jpn. J. Cancer Res., 1986, 79, 866-73). Other studies usingantisense oligonucleotides containing CpG dinucleotides have been shownto stimulate immune responses (Zhao Q, et al. (1996) Biochem.Pharmacol.26:173-182). Subsequent studies demonstrated that TLR9 recognizesunmethylated CpG motifs present in bacterial and synthetic DNA (Hemmi,H. et al. (2000) Nature 408:740-745). Other modifications ofCpG-containing phosphorothioate oligonucleotides can also affect theirability to act as modulators of immune response through TLR9 (see, e.g.,Zhao et al., Biochem. Pharmacol. (1996) 51:173-182; Zhao et al. (1996)Biochem Pharmacol. 52:1537-1544; Zhao et al. (1997) Antisense NucleicAcid Drug Dev. 7:495-502; Zhao et al (1999) Bioorg. Med. Chem. Lett.9:3453-3458; Zhao et al. (2000) Bioorg. Med. Chem. Lett. 10:1051-1054;Yu, D. et al. (2000) Bioorg. Med. Chem. Lett. 10:2585-2588; Yu, D. etal. (2001) Bioorg. Med. Chem. Lett. 11:2263-2267; and Kandimalla, E. etal. (2001) Bioorg. Med. Chem. 9:807-813). In addition, structureactivity relationship studies have allowed identification of syntheticmotifs and novel DNA-based compounds that induce specific immuneresponse profiles that are distinct from those resulting fromunmethylated CpG dinucleotides. (Kandimalla, E. et al. (2005) Proc.Natl. Acad. Sci. USA 102:6925-6930. Kandimalla, E. et al. (2003) Proc.Nat. Acad. Sci. USA 100:14303-14308; Cong, Y. et al. (2003) BiochemBiophys Res. Commun.310:1133-1139; Kandimalla, E. et al. (2003) Biochem.Biophys. Res. Commun. 306:948-953; Kandimalla, E. et al. (2003) NucleicAcids Res. 31:2393-2400; Yu, D. et al. (2003) Bioorg. Med. Chem.11:459-464; Bhagat, L. et al. (2003) Biochem. Biophys. Res. Commun.300:853-861; Yu, D. et al. (2002) Nucleic Acids Res. 30:4460-4469; Yu,D. et al. (2002) J. Med. Chem.45:4540-4548. Yu, D. et al. (2002)Biochem. Biophys. Res. Commun.297:83-90; Kandimalla. E. et al. (2002)Bioconjug. Chem.13:966-974; Yu, D. et al. (2002) Nucleic Acids Res.30:1613-1619; Yu, D. et al. (2001) Bioorg. Med. Chem. 9:2803-2808; Yu,D. et al. (2001) Bioorg. Med. Chem. Lett. 11:2263-2267; Kandimalla, E.et al. (2001) Bioorg. Med. Chem. 9:807-813; Yu, D. et al. (2000) Bioorg.Med. Chem. Lett. 10:2585-2588; Putta, M. et al. (2006) Nucleic AcidsRes. 34:3231-3238).

The selective localization of TLRs and the signaling generatedtherefrom, provides some insight into their role in the immune response.The immune response involves both an innate and an adaptive responsebased upon the subset of cells involved in the response. For example,the T helper (Th) cells involved in classical cell-mediated functionssuch as delayed-type hypersensitivity and activation of cytotoxic Tlymphocytes (CTLs) are Th1 cells. This response is the body's innateresponse to antigen (e.g. viral infections, intracellular pathogens, andtumor cells), and results in a secretion of IFN-gamma and a concomitantactivation of CTLs. Alternatively, the Th cells involved as helper cellsfor B-cell activation are Th2 cells. Th2 cells have been shown to beactivated in response to bacteria and parasites and may mediate thebody's adaptive immune response (e.g. IgE production and eosinophilactivation) through the secretion of IL-4 and IL-5. The type of immuneresponse is influenced by the cytokines produced in response to antigenexposure and the differences in the cytokines secreted by Th1 and Th2cells may be the result of the different biological functions of thesetwo subsets.

While activation of TLRs is involved in mounting an immune response, anuncontrolled stimulation of the immune system through TLRs mayexacerbate certain diseases in immune compromised subjects. In recentyears, several groups have shown the use of syntheticoligodeoxyoligonucleotides (ODNs) as inhibitors of inflammatorycytokines (Lenert, P. et al. (2003) DNA Cell Biol. 22(10):621-631).

Using certain synthetic ODNs, Lenert et al. report the ability toproduce inhibitory ODNs (Lenert, P. et al. (2003) DNA Cell Biol.22(10):621-631). These inhibitory ODN require two triplet sequences, aproximal “CCT” triplet and a distal “GGG” triplet. In addition to thesetriplet-containing inhibitory ODNs, several groups have reported otherspecific DNA sequences that could inhibit TLR-9-mediated activation byCpG-containing ODNs. These “inhibitory” or “suppressive” motifs are richin poly “G” (e.g. “GGGG”) or “GC” sequences, tend to be methylated, andare present in the DNA of mammals and certain viruses (see e.g.,; Chen,Y., et al., Gene Ther. 8: 1024-1032 (2001); Stunz, L. L., Eur. J.Immunol. 32: 1212-1222 (2002). Duramad, O., et al., J. Immunol., 174:5193-5200 (2005) and Jurk et. al (U.S. 2005/0239733), describe astructure for inhibitory DNA oligonucleotides containing a GGGG motifwithin the sequences. Patole et al. demonstrate that GGGG containingODNs will suppress systemic lupus (Patole, P. et al. (2005) J. Am. Soc.Nephrol. 16:3273-3280). Additionally, Gursel, I., et al., J. Immunol.,171: 1393-1400 (2003), describe repetitive TTAGGG elements, which arepresent at high frequency in mammalian telomeres, down-regulateCpG-induced immune activation. Shirota, H., et al., J. Immunol., 173:5002-5007 (2004), demonstrate that synthetic oligonucleotides containingthe TTAGGG element mimic this activity and could be effective in theprevention/treatment of certain Th1-dependent autoimmune diseases.

In contrast, recent studies have called into question the view that polyG containing ODNs are acting as antagonists of TLRs. For example, U.S.Pat. No. 6,426,334, Agrawal et al., demonstrate that administering CpGoligonucleotides containing GGGG strings have potent antiviral andanticancer activity, and further that administration of these compoundswill cause an increase in serum IL-12 concentration. Further, CpG oligoscontaining polyG sequences are known to induce immune responses throughTLR9 activation (Verthelyi D et al, J Immunol. 166, 2372, 2001; Gursel Met al, J Leukoc Biol, 71, 813, 2001, Krug A et al, Eur J Immunol, 31,2154, 2001) and show antitumor, antiviral activities (Ballas GK et al, JImmunol, 167, 4878, 2001; Verthelyi D et al, J Immunol, 170, 4717,2003). In addition, polyG oligonucleotides are also known to inhibit HIVand Rel A (McShan W M, et al, J Biol Chem., 267(8):5712-21, 1992; Rando,R F et al., J Biol Chem, 270(4):1754-60, 1995; Benimetskaya L, et al.,Nucleic Acids Res., 25(13):2648-56, 1997). In addition, ODNs containingan immune stimulatory CpG motif and 4 consecutive G nucleotides (class AODNs) induce interferon-γ production and a Th1 shift in the immuneresponse. Moreover, in preclinical disease models, Class A ODN have beenshown to induce a TLR-mediated immune response.

In addition, oligonucleotides containing guanosine strings have beenshown to form tetraplex structures, act as aptamers and inhibit thrombinactivity (Bock LC et al., Nature, 355:564-6, 1992; Padmanabhan, K etal., J Biol Chem., 268(24):17651-4, 1993). Thus it is not clear whethersingle-stranded or multiple-stranded structures are effective atsuppressing TLR9 activation.

Thus, there is a need for effective antagonist of TLRs without a concernthat they will form secondary structures.

BRIEF SUMMARY OF THE INVENTION

The invention provides novel immune regulatory oligonucleotides (IRO)compounds as antagonists of TLRs and methods of use thereof. These IROshave one or more chemical modifications in the sequence flanking animmune stimulatory motif and/or in an oligonucleotide motif that wouldbe immune stimulatory but for the modification.

The invention further provides novel IRO compositions having thestructure 5-N_(m)—N₃N₂N₁CGN¹N²N³—N^(m-)3′, wherein CG is anoligonucleotide motif and C is cytosine or a pyrimidine nucleotidederivative or non-nucleotide linkage, and G is guanosine a purinenucleotide derivative or non-nucleotide linkage; N1-N3, at eachoccurrence, is independently a nucleotide, nucleotide derivative ornon-nucleotide linkage; Nm, at each occurrence, is independently anucleotide, nucleotide derivative or non-nucleotide linkage; providedthat at least one N1 to N3 and/or C and/or G is a nucleotide derivativeor non-nucleotide linkage; and further provided that compound containsless than 4 consecutive guanosine nucleotides wherein theoligonucleotide motif would be immune stimulatory but for the nucleotidederivative or non-nucleotide linkage; and wherein m is a number from 0to about 30.

The invention further provides for a pharmaceutical compositioncomprising any an IRO and a pharmaceutically acceptable carrier.

The invention provides for a method for modifying a TLR-stimulatingoligonucleotide comprising an immune stimulatory oligonucleotide motifcomprising incorporating chemical modifications into the immunestimulatory oligonucleotide motif and/or to the sequence flanking theimmune stimulatory oligonucleotide motif, wherein the immune stimulatoryactivity of the immune stimulatory oligonucleotide motif is suppressedby the chemical modifications.

The invention further provides a method for inhibiting a TLR-mediatedimmune response in a vertebrate, the method comprising administering tothe vertebrate an IRO compound in a pharmaceutically effective amount,wherein the route of administration is parenteral, mucosal delivery,oral, sublingual, transdermal, topical, inhalation, intranasal, aerosol,intraocular, intratracheal, intrarectal, vaginal, by gene gun, dermalpatch or in eye drop or mouthwash form. In some preferred embodiments,inhibiting TLR stimulation comprising administering an IRO compoundaccording to the invention, wherein the TLR is selected from TLR2, TLR3,TLR4, TLR5, TLR7, TLR8, and TLR9.

The invention further provides a method for inhibiting the activity of aTLR agonist comprising administering an IRO compound, wherein the IRO isadministered at the same time, prior to or after the TLR agonist. Inpreferred embodiments the TLR agonist is selected from an agonist ofTLR2, TLR3, TLR4, TLR5, TLR7, TLR8, and TLR9.

The invention further provides a method for therapeutically treating avertebrate having a disease mediated by a TLR, such method comprisingadministering to the vertebrate an IRO compound according to theinvention in a pharmaceutically effective amount. In preferredembodiments, the disease is cancer, an autoimmune disorder, airwayinflammation, inflammatory disorders, infectious disease, skindisorders, allergy, asthma or a disease caused by a pathogen.

In some preferred embodiments, the IRO compound is administered incombination with one or more vaccines, antigens, antibodies, cytotoxicagents, allergens, antibiotics, antisense oligonucleotides, TLRagonists, TLR antagonists, peptides, proteins, gene therapy vectors, DNAvaccines, adjuvants or co-stimulatory molecules. In some preferredembodiments, the route of administration is parenteral, mucosaldelivery, oral, sublingual, transdermal, topical, inhalation,intranasal, aerosol, intraocular, intratracheal, intrarectal, vaginal,by gene gun, dermal patch or in eye drop or mouthwash form.

The invention further provides a method for preventing cancer, anautoimmune disorder, airway inflammation, inflammatory disorders,infectious disease, skin disorders, allergy, asthma or a disease causedby a pathogen in a vertebrate, such method comprising administering tothe vertebrate an IRO compound according to the invention in apharmaceutically effective amount. Preferred autoimmune disordersinclude without limitation lupus, multiple sclerosis, type I diabetesmellitus, irritable bowl syndrome, Chron's disease, arthritis, septicshock and malaria.

In some preferred embodiments, the IRO compound is administered incombination with one or more vaccines, antigens, antibodies, cytotoxicagents, allergens, antibiotics, antisense oligonucleotides, TLRagonists, TLR antagonists, peptides, proteins, gene therapy vectors, DNAvaccines, adjuvants or co-stimulatory molecules. In some preferredembodiments, the route of administration is parenteral, mucosaldelivery, oral, sublingual, transdermal, topical, inhalation,intranasal, aerosol, intraocular, intratracheal, intrarectal, vaginal,by gene gun, dermal patch or in eye drop or mouthwash form.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates IRO inhibition of the TLR9 agonist activity of anIMO (SEQ ID NOS 1, 5, and 6).

FIG. 2 demonstrates the specificity of one IRO compound as an antagonistof TLR9 vs TLR3 (SEQ ID NOS 1 and 5).

FIG. 3 demonstrates dose-dependent inhibition by an IRO (SEQ ID NOS 3and 5).

FIGS. 4A-4D demonstrate that pre-administration and simultaneousadministration of IRO can inhibit an agonist of TLR9 (SEQ ID NOS 3-5).

FIGS. 5A and 5B demonstrate that two CpG oligonucleotides linked attheir 5′ ends show TLR-inhibitory properties (SEQ ID NOS 3, 21, and 4).

FIGS. 6A and 6B demonstrate that an IRO inhibited TLR9 agonist activityin human cell cultures (SEQ ID NOS 2 and 10).

FIGS. 7A through 7D demonstrate an IRO effect on OVA induced Th2 and Th1immune responses (SEQ ID NOS 1 and 5).

FIGS. 8A through 8D demonstrates that an IRO reversed Th2 inhibitoryproperties and inhibited Th1 immune responses induced by an IMO (SEQ IDNOS 1 and 5).

FIGS. 9A through 9C demonstrates antibody responses to an IMO and an IRO(SEQ ID NOS 1, 5, and 6).

FIGS. 10A through 10C demonstrates early inhibitory activity of selectedIROs on TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, and TLR9 in vivo.

FIG. 11 demonstrates early inhibitory activity of selected IROs on TLR2,TLR3, TLR4, TLR5, TLR7, TLR8, and TLR9 in vivo.

FIG. 12 demonstrates early inhibitory activity of selected IROs on TLR2,TLR3, TLR4, TLR5, TLR7, TLR8, and TLR9 in vivo.

FIG. 13 demonstrates long-term antagonist activity of selected IROs onTLR2, TLR3, TLR4, TLR5, TLR7, TLR8, and TLR9 in vivo.

FIG. 14 demonstrates long-term antagonist activity of selected IROs onTLR2, TLR3, TLR4, TLR5, TLR7, TLR8, and TLR9 in vivo.

FIG. 15 demonstrates long-term antagonist activity of selected IROs onTLR2, TLR3, TLR4, TLR5, TLR7, TLR8, and TLR9 in vivo.

FIG. 16 demonstrates that an IRO inhibits proliferation of wild type(BALB/c) and lupus prone (MRL-lpr) mice B lymphocyte proliferation invitro.

FIGS. 17A through 17F demonstrate that an IRO inhibited IL-6 and IL-12production by wild type (BALB/c) and lupus prone (MRL-lpr) mice Blymphocytes and lupus prone (NZBW) mice spleen cells in vitro.

FIGS. 18A through 18F demonstrate that MRL-lpr mice injected with an IROreduced levels of anti-DNA IgG1 and IgG2a antibodies in serum andprotein in urine.

FIG. 19 demonstrates that an IRO inhibits serum anti-DNA IgG2a in NZBWmice.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to the therapeutic use of noveloligonucleotides as immune modulatory agents for immunotherapyapplications. Specifically, the invention provides Immune RegulatoryOligonucleotide (IRO) compounds as antagonists of toll-like receptors(TLRs) to inhibit and/or suppress a TLR-mediated immune response. TheseIROs have unique sequences that inhibit or suppress TLR-mediatedsignaling in response to endogenous and/or exogenous TLR ligands oragonists. The references cited herein reflect the level of knowledge inthe field and are hereby incorporated by reference in their entirety.Any conflicts between the teachings of the cited references and thisspecification shall be resolved in favor of the latter.

The invention provides methods for suppressing an immune response causedby TLRs and can be used for immunotherapy applications such as, but notlimited to, treatment of cancer, autoimmune disorders, asthma,respiratory allergies, food allergies, skin allergies, systemic lupuserythematosus (SLE), arthritis, pleurisy, chronic infections,inflammatory diseases, inflammatory bowl syndrome, sepsis, and bacteria,parasitic, and viral infections in adult and pediatric human andveterinary applications. Thus, the invention further provides IROcompounds having optimal levels of immune modulatory effect forimmunotherapy and methods for making and using such compounds. Inaddition, IRO compounds of the invention are useful in combination with,for example, DNA vaccines, antigens, antibodies, and allergens; and incombination with chemotherapeutic agents (both traditional chemotherapyand modern targeted therapies) and/or antisense oligonucleotides forprevention and treatment of diseases.

Definitions

The term “oligonucleotide” generally refers to a polynucleosidecomprising a plurality of linked nucleoside units. Such oligonucleotidescan be obtained from existing nucleic acid sources, including genomic orcDNA, but are preferably produced by synthetic methods. In preferredembodiments each nucleoside unit can encompass various chemicalmodifications and substitutions as compared to wild-typeoligonucleotides, including but not limited to modified nucleoside baseand/or modified sugar unit. Examples of chemical modifications are knownto the person skilled in the art and are described, for example, inUhlmann E et al. (1990) Chem. Rev. 90:543; “Protocols forOligonucleotides and Analogs” Synthesis and Properties & Synthesis andAnalytical Techniques, S. Agrawal, Ed, Humana Press, Totowa, USA 1993;and Hunziker, J. et al. (1995) Mod. Syn. Methods 7:331-417; and Crooke,S. et al. (1996) Ann.Rev. Pharm. Tox. 36:107-129. The nucleosideresidues can be coupled to each other by any of the numerous knownintemucleoside linkages. Such internucleoside linkages include, withoutlimitation, phosphodiester, phosphorothioate, phosphorodithioate,alkylphosphonate, alkylphosphonothioate, phosphotriester,phosphoramidate, siloxane, carbonate, carboalkoxy, acetamidate,carbamate, morpholino, borano, thioether, bridged phosphoramidate,bridged methylene phosphonate, bridged phosphorothioate, and sulfoneinternucleoside linkages. The term “oligonucleotide” also encompassespolynucleosides having one or more stereospecific internucleosidelinkage (e.g., (R_(P))- or (S_(P))-phosphorothioate, alkylphosphonate,or phosphotriester linkages). As used herein, the terms“oligonucleotide” and “dinucleotide” are expressly intended to includepolynucleosides and dinucleosides having any such internucleosidelinkage, whether or not the linkage comprises a phosphate group. Incertain preferred embodiments, these internucleoside linkages may bephosphodiester, phosphorothioate, or phosphorodithioate linkages, orcombinations thereof.

The term “2′-substituted ribonucleoside” or “2′-substituted arabinoside”generally includes ribonucleosides or arabinonucleosides in which thehydroxyl group at the 2′ position of the pentose moiety is substitutedto produce a 2′-substituted or 2′-O-substituted ribonucleoside. Incertain embodiments, such substitution is with a lower hydrocarbyl groupcontaining 1-6 saturated or unsaturated carbon atoms, with a halogenatom, or with an aryl group having 6-10 carbon atoms, wherein suchhydrocarbyl, or aryl group may be unsubstituted or may be substituted,e.g., with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy,alkoxy, carboxyl, carboalkoxy, or amino groups. Examples of2′-O-substituted ribonucleosides or 2′-O-substituted-arabinosidesinclude, without limitation 2′-amino, 2′-fluoro, 2′-allyl, 2′-O-alkyland 2′-propargyl ribonucleosides or arabinosides,2′-O-methylribonucleosides or 2′-O-methylarabinosides and2′-O-methoxyethoxyribonucleosides or 2′-O-methoxyethoxyarabinosides.

The term “3′”, when used directionally, generally refers to a region orposition in a polynucleotide or oligonucleotide 3′ (downstream) fromanother region or position in the same polynucleotide oroligonucleotide.

The term “5′”, when used directionally, generally refers to a region orposition in a polynucleotide or oligonucleotide 5′ (upstream) fromanother region or position in the same polynucleotide oroligonucleotide.

The term “about” generally means that the exact number is not critical.Thus, the number of nucleoside residues in the oligonucleotides is notcritical, and oligonucleotides having one or two fewer nucleosideresidues, or from one to several additional nucleoside residues arecontemplated as equivalents of each of the embodiments described above.

The term “agonist” generally refers to a substance that binds to areceptor of a cell and induces a response. An agonist often mimics theaction of a naturally occurring substance such as a ligand.

The term “antagonist” generally refers to a substance that attenuatesthe effects of an agonist.

The term “adjuvant” generally refers to a substance which, when added toan immunogenic agent such as vaccine or antigen, enhances or potentiatesan immune response to the agent in the recipient host upon exposure tothe mixture.

The term “airway inflammation” generally includes, without limitation,asthma.

The term “allergen” generally refers to an antigen or antigenic portionof a molecule, usually a protein, which elicits an allergic responseupon exposure to a subject. Typically the subject is allergic to theallergen as indicated, for instance, by the wheal and flare test or anymethod known in the art. A molecule is said to be an allergen even ifonly a small subset of subjects exhibit an allergic immune response uponexposure to the molecule.

The term “allergy” generally refers to an inappropriate immune responsecharacterized by inflammation and includes, without limitation, foodallergies and respiratory allergies.

The term “antigen” generally refers to a substance that is recognizedand selectively bound by an antibody or by a T cell antigen receptor,resulting in induction of an immune response. Antigens may include butare not limited to peptides, proteins, nucleosides, nucleotides, andcombinations thereof. Antigens may be natural or synthetic and generallyinduce an immune response that is specific for that antigen.

The term “autoimmune disorder” generally refers to disorders in which“self” components undergo attack by the immune system.

The term “TLR-mediated disease” or TLR-mediated disorder” generallymeans any pathological condition for which activation of one or moreTLRs is a contributing factor. Such conditions include but are notlimited, cancer, an autoimmune disorder, airway inflammation,inflammatory disorders, infectious disease, skin disorders, allergy,asthma or a disease caused by a pathogen.

The term “physiologically acceptable” generally refers to a materialthat does not interfere with the effectiveness of an IRO compound andthat is compatible with a biological system such as a cell, cellculture, tissue, or organism. Preferably, the biological system is aliving organism, such as a vertebrate.

The term “carrier” generally encompasses any excipient, diluent, filler,salt, buffer, stabilizer, solubilizer, oil, lipid, lipid containingvesicle, microspheres, liposomal encapsulation, or other material wellknown in the art for use in pharmaceutical formulations. It will beunderstood that the characteristics of the carrier, excipient, ordiluent will depend on the route of administration for a particularapplication. The preparation of pharmaceutically acceptable formulationscontaining these materials is described in, e.g., Remington'sPharmaceutical Sciences, 18th Edition, ed. A. Gennaro, Mack PublishingCo., Easton, Pa., 1990.

The term “co-administration” generally refers to the administration ofat least two different substances sufficiently close in time to modulatean immune response. Co-administration refers to simultaneousadministration, as well as temporally spaced order of up to several daysapart, of at least two different substances in any order, either in asingle dose or separate doses.

The term “complementary” generally means having the ability to hybridizeto a nucleic acid. Such hybridization is ordinarily the result ofhydrogen bonding between complementary strands, preferably to formWatson-Crick or Hoogsteen base pairs, although other modes of hydrogenbonding, as well as base stacking can also lead to hybridization.

The term an “effective amount” or a “sufficient amount” generally refersto an amount sufficient to affect a desired biological effect, such asbeneficial results. Thus, an “effective amount” or “sufficient amount”will depend upon the context in which it is being administered. In thecontext of administering a composition that modulates an immune responseto a co-administered antigen, an effective amount of an IRO compound andantigen is an amount sufficient to achieve the desired modulation ascompared to the immune response obtained when the antigen isadministered alone. An effective amount may be administered in one ormore administrations.

The term “in combination with” generally means in the course of treatinga disease or disorder in a patient, administering an IRO compound and anagent useful for treating the disease or disorder that does not diminishthe immune modulatory effect of the IRO compound. Such combinationtreatment may also include more than a single administration of an IROcompound and/or independently an agent. The administration of the IROcompound and/or the agent may be by the same or different routes.

The term “individual” or “subject” or “vertebrate” generally refers to amammal, such as a human. Mammals generally include, but are not limitedto, humans, non-human primates, rats, mice, cats, dogs, horses, cattle,cows, pigs, sheep, and rabbits.

The term “nucleoside” generally refers to compounds consisting of asugar, usually ribose or deoxyribose, and a purine or pyrimidine base.

The term “nucleotide” generally refers to a nucleoside comprising aphosphate group attached to the sugar.

As used herein, the term “pyrimidine nucleoside” refers to a nucleosidewherein the base component of the nucleoside is a pyrimidine base (e.g.,cytosine (C) or thymine (T) or Uracil (U)). Similarly, the term “purinenucleoside” refers to a nucleoside wherein the base component of thenucleoside is a purine base (e.g., adenine (A) or guanine (G)).

The terms “analog” or “derivative” can be used interchangeable togenerally refer to any purine and/or pyrimidine nucleotide or nucleosidethat has a modified base and/or sugar. A modified base is a base that isnot guanine, cytosine, adenine, thymine or uracil. A modified sugar isany sugar that is not ribose or 2′deoxyribose and can be used in thebackbone for an oligonucleotide.

The term “inhibiting” or “suppressing” generally refers to a decrease ina response or qualitative difference in a response, which couldotherwise arise from eliciting and/or stimulation of a response.

The term “non-nucleotide linker” generally refers to any linkage ormoiety that can link or be linked to the oligonucleotides other thanthrough a phosphorous-containing linkage. Preferably such linker is fromabout 2 angstroms to about 200 angstroms in length.

The term “nucleotide linkage” generally refers to a direct 3′-5′ linkagethat directly connects the 3′ and 5′ hydroxyl groups of two nucleosidesthrough a phosphorous-containing linkage.

The terms “oligonucleotide motif” means an oligonucleotide sequence,including a dinucleotide. An “oligonucleotide motif that would be immunestimulatory, but for one or more modifications” means an oligonucleotidemotif which is immune stimulatory in a parent oligonucleotide, but notin a derivative oligonucleotide, wherein the derivative oligonucleotideis based upon the parent oligonucleotide, but has one or moremodifications.

The terms CpG, C*pG, C*pG* and CpG* refer to oligonucleotide motifs thatare immune stimulatory and comprise cytosine or a cytosine analog and aguanine or a guanine analog.

The term “treatment” generally refers to an approach intended to obtaina beneficial or desired results, which may include alleviation ofsymptoms, or delaying or ameliorating a disease progression.

In a first aspect, the invention provides an immune regulatoryoligonucleotide (IRO) compound. The term “IRO” refers to an immuneregulatory oligonucleotide compound that is an antagonist for one ormore TLR, wherein the compound comprises an oligonucleotide motif and atleast one modification, wherein the oligonucleotide motif would beimmune stimulatory (e.g., unmethylated CpG), but for the one or moremodifications that suppress the activity of the oligonucleotide motif,provided that compound contains less than 4 consecutive guanosinenucleotides and preferably less than 3 consecutive guanosinenucleotides. Such modifications may be in the oligonucleotide 5′terminus, in a sequence flanking the oligonucleotide motif, and/orwithin the oligonucleotide motif. These modifications result in an IROcompound that suppresses TLR-modulated immune stimulation. Suchmodifications can be to the bases, sugar residues and/or the phosphatebackbone of the nucleotides/nucleosides flanking the oligonucleotidemotif or within the oligonucleotide motif.

In preferred embodiments, when the modification is a 2′ alkylation oralkoxylation then the modification is not 5′ adjacent to theoligonucleotide motif, when the modification is a non-chargedinternucleoside linkage then the modification is not 5′ adjacent to theoligonucleotide motif, and when the modification is a 3′ alkylation oralkoxylation then the modification is not 5′ or 3′ adjacent to theoligonucleotide motif.

In preferred embodiments the IRO compound is not an antisenseoligonucleotide.

The general structure of the IRO compounds may be represented as5′-N_(m)—N₃N₂N₁CGN¹N²N³—N^(m-)3′ wherein CG is an immune stimulatorymotif and C is cytosine or a pyrimidine nucleotide derivative ornon-nucleotide linker, and G is guanosine, a purine nucleotidederivative or non-nucleotide linker; N1-N3, at each occurrence, isindependently a nucleotide, nucleotide derivative or non-nucleotidelinker; Nm, at each occurrence, is independently a nucleotide,nucleotide derivative or non-nucleotide linker; provided that at leastone N1 to N3 and/or C and/or G is a nucleotide derivative ornon-nucleotide linker; and further provided that compound contains lessthan 4 consecutive guanosine nucleotides and preferably less than 3consecutive guanosines, wherein the immune stimulatory activity of theCG is suppressed by the nucleotide derivative or non-nucleotide linker;and wherein m is a number from 0 to about 30.

In certain embodiments of the invention, IRO compounds may comprise atleast two oligonucleotides covalently linked by a nucleotide linkage, ora non-nucleotide linker, at their 5′-, 3′- or 2′-ends or byfunctionalized sugar or by functionalized nucleobase via anon-nucleotide linker or a nucleotide linkage. Such IRO compounds may belinear or branched. As a non-limiting example, the linker may beattached to the 3′-hydroxyl. In such embodiments, the linker comprises afunctional group, which is attached to the 3′-hydroxyl by means of aphosphate-based linkage like, for example, phosphodiester,phosphorothioate, phosphorodithioate, methylphosphonate, or bynon-phosphate-based linkages. Possible sites of conjugation for theribonucleotide are indicated in Formula I, below, wherein B represents aheterocyclic base and wherein the arrow pointing to P indicates anyattachment to phosphorous.

In some embodiments, the non-nucleotide linker is a small molecule,macromolecule or biomolecule, including, without limitation,polypeptides, antibodies, lipids, antigens, allergens, andoligosaccharides. In some other embodiments, the non-nucleotidic linkeris a small molecule. For purposes of the invention, a small molecule isan organic moiety having a molecular weight of less than 1,000 Da. Insome embodiments, the small molecule has a molecular weight of less than750 Da.

In some embodiments, the small molecule is an aliphatic or aromatichydrocarbon, either of which optionally can include, either in thelinear chain connecting the oligoribonucleotides or appended to it, oneor more functional groups including, but not limited to, hydroxy, amino,thiol, thioether, ether, amide, thioamide, ester, urea, or thiourea. Thesmall molecule can be cyclic or acyclic. Examples of small moleculelinkers include, but are not limited to, amino acids, carbohydrates,cyclodextrins, adamantane, cholesterol, haptens and antibiotics.However, for purposes of describing the non-nucleotidic linker, the term“small molecule” is not intended to include a nucleoside.

In some embodiments, the non-nucleotidic linker is an alkyl linker oramino linker. The alkyl linker may be branched or unbranched, cyclic oracyclic, substituted or unsubstituted, saturated or unsaturated, chiral,achiral or racemic mixture. The alkyl linkers can have from about 2 toabout 18 carbon atoms. In some embodiments such alkyl linkers have fromabout 3 to about 9 carbon atoms. Some alkyl linkers include one or morefunctional groups including, but not limited to, hydroxy, amino, thiol,thioether, ether, amide, thioamide, ester, urea, and thioether. Suchalkyl linkers can include, but are not limited to, 1,2 propanediol,1,2,3 propanetriol, 1,3 propanediol, triethylene glycol hexaethyleneglycol, polyethylene glycollinkers (e.g. [—O—CH2-CH2-]_(n)(n=1-9)),methyl linkers, ethyl linkers, propyl linkers, butyl linkers,or hexyl linkers. In some embodiments, such alkyl linkers may includepeptides or amino acids.

In some embodiments, the non-nucleotide linker may include, but are notlimited to, those listed in Table 2.

TABLE 2 Representative Non-Nucleotidic Linkers

In some embodiments, the small molecule linker is glycerol or a glycerolhomolog of the formula HO—(CH₂)_(o)—CH(OH)—(CH₂)_(p)—OH, wherein o and pindependently are integers from 1 to about 6, from 1 to about 4, or from1 to about 3. In some other embodiments, the small molecule linker is aderivative of 1,3-diamino-2-hydroxypropane. Some such derivatives havethe formula HO—(CH₂)_(m)—C(O)NH—CH₂—CH(OH)—CH₂—NHC(O)—(CH₂)_(m)—OH,wherein m is an integer from 0 to about 10, from 0 to about 6, from 2 toabout 6, or from 2 to about 4

Some non-nucleotide linkers according to the invention permit attachmentof more than two oligonucleotides. For example, the small moleculelinker glycerol has three hydroxyl groups to which oligonucleotides maybe covalently attached. Some IROs according to the invention, therefore,comprise two or more oligonucleotides linked to a nucleotide or anon-nucleotide linker. Such IROs are referred to as being “branched”.

IRO compounds may comprise at least two oligonucleotides non-covalentlylinked, such as by electrostatic interactions, hydrophobic interactions,π-stacking interactions, hydrogen bonding and combinations thereof.Non-limiting examples of such non-covalent linkage includes Watson-Crickbase pairing, Hoogsteen base pairing and base stacking.

Some of the ways in which two or more oligonucleotides can be linked areshown in Table 3.

TABLE 3 Oligoribonucleotide Formulas IV-XI Formula IV

Formula V

Formula VI

Formula VII

Formula VIII

Formula IX

Formula X

Formula XI

In certain embodiments, pyrimidine nucleosides in the immune regulatoryoligonucleotides used in the compositions and methods according to theinvention have the structure (II):

wherein:

D is a hydrogen bond donor;

D′ is selected from the group consisting of hydrogen, hydrogen bonddonor, hydrogen bond acceptor, hydrophilic group, hydrophobic group,electron withdrawing group and electron donating group;

A is a hydrogen bond acceptor or a hydrophilic group;

A′ is selected from the group consisting of hydrogen bond acceptor,hydrophilic group, hydrophobic group, electron withdrawing group andelectron donating group;

X is carbon or nitrogen; and

S′ is a pentose or hexose sugar ring, or a sugar analog.

In certain embodiments, the sugar ring is derivatized with a phosphatemoiety, modified phosphate moiety, or other linker moiety suitable forlinking the pyrimidine nucleoside to another nucleoside or nucleosideanalog.

In some embodiments hydrogen bond donors include, without limitation,—NH—, —NH₂, —SH and —OH. Preferred hydrogen bond acceptors include,without limitation, C═O, C═S, and the ring nitrogen atoms of an aromaticheterocycle, e.g., N3 of cytosine.

In some embodiments, (II) is a pyrimidine nucleoside derivative.Examples of pyrimidine nucleoside derivatives include, withoutlimitation, 5-hydroxycytosine, 5-hydroxymethylcytosine,N4-alkylcytosine, or N4-ethylcytosine, araC, 5-OH-dC, N3-Me-dC, and4-thiouracil. Chemical modified derivatives also include, but are notlimited to, thymine or uracil analogues. In some embodiments, the sugarmoiety S′ in (II) is a sugar derivative. Suitable sugar derivativesinclude, but are not limited to, trehalose or trehalose derivatives,hexose or hexose derivatives, arabinose or arabinose derivatives.

In some embodiments, the purine nucleosides in immune regulatoryoligonucleotides used in the compositions and methods according to theinvention have the structure (III):

wherein:

D is a hydrogen bond donor;

D′ is selected from the group consisting of hydrogen, hydrogen bonddonor, and hydrophilic group;

A is a hydrogen bond acceptor or a hydrophilic group;

X is carbon or nitrogen;

each L is independently selected from the group consisting of C, O, Nand S; and

S′ is a pentose or hexose sugar ring, or a sugar analog.

In certain embodiments, the sugar ring is derivatized with a phosphatemoiety, modified phosphate moiety, or other linker moiety suitable forlinking the pyrimidine nucleoside to another nucleoside or nucleosideanalog.

In certain embodiments hydrogen bond donors include, without limitation,—NH—, —NH₂, —SH and —OH. In certain embodiments hydrogen bond acceptorsinclude, without limitation, C═O, C═S, —NO₂ and the ring nitrogen atomsof an aromatic heterocycle, e.g., N1 of guanine.

In some embodiments, (III) is a purine nucleoside derivative. Examplesof purine nucleoside derivatives include, without limitation, guanineanalogues such as 7-deaza-G, 7-deaza-dG, ara-G, 6-thio-G, Inosine,Iso-G, loxoribine, TOG(7-thio-8-oxo)-G, 8-bromo-G, 8-hydroxy-G,5-aminoformycin B, Oxoformycin, 7-methyl-G, 9-p-chlorophenyl-8-aza-G,9-phenyl-G, 9-hexyl-guanine, 7-deaza-9-benzyl-G,6-Chloro-7-deazaguanine, 6-methoxy-7-deazaguanine, 8-Aza-7-deaza-G(PPG),2-(Dimethylamino)guanosine, 7-Methyl-6-thioguanosine,8-Benzyloxyguanosine, 9-Deazaguanosine,1-(B-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine,1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine. Chemicallymodified derivatives also include, but are not limited to, adenineanalogues such as 9-benzyl-8-hydroxy-2-(2-methoxyethoxy)adenine,2-Amino-N2-O—, methyladenosine, 8-Aza-7-deaza-A, 7-deaza-A, Vidarabine,2-Aminoadenosine, N1-Methyladenosine, 8-Azaadenosine, 5-Iodotubercidin,and N1-Me-dG. In some embodiments, the sugar moiety S′ in (III) is asugar derivative as defined for Formula II.

In certain embodiments of the invention, the immune regulatory nucleicacid comprises a nucleic acid sequence containing at least one B-L-deoxynucleoside or 3′-deoxy nucleoside.

In certain embodiments of the invention, the immune regulatoryoligonucleotide comprises a nucleic acid sequence containing at leastone dinucleotide selected from CpG, C*pG, C*pG* and CpG*, wherein C iscytosine or 2′-deoxycytidine, G is guanosine or 2′-deoxyguanosine, C* is2′-deoxythymidine,1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine,2′-dideoxy-5-halocytosine, 2′-dideoxy-5-nitrocytosine, arabinocytidine,2′-deoxy-2′-substituted arabinocytidine, 2′-O-substitutedarabinocytidine, 2′-deoxy-5-hydroxycytidine, 2′-deoxy-N4-alkyl-cytidine,2′-deoxy-4-thiouridine, or other pyrimidine nucleoside analogs, G* is2′-deoxy-7-deazaguanosine, 2′-deoxy-6-thioguanosine, arabinoguanosine,2′-deoxy-2′substituted-arabinoguanosine,2′-O-substituted-arabinoguanosine, 2′-deoxyinosine, or other purinenucleoside analogs, and p is an internucleoside linkage selected fromthe group consisting of phosphodiester, phosphorothioate, andphosphorodithioate, and wherein the activity of the at least onedinucleotide is regulated by the flanking sequence.

The sequences of specific IRO within these general structures used inthe present study include, but are not limited to, those shown in Table4.

TABLE 4 IRO/SEQ ID NO: Sequence 5 5′-CTATCTGACGTTCTCTGT-3′ 75′-CTATCTGACGTTCTCTGT-3′ 17 5′-CTATCTGACG₁TTCTCTGT-3′ 375′-CTATCTGACG₄TTCTCTGT-3′ 39 5′-CTATCTGAC₄GTTCTCTGT-3′ 415′-CTATCTGAC₅GTTCTCTGT-3′ 43 5′-CTATCTGAC₆GTTCTCTGT-3′ 455′-CTATCTGACG₅TTCTCTGT-3′ 47 5′-CTATCTGAC₇GTTCTCTGT-3′ 645′-CTATCTAACGTTCTCTGT-3′ 67 5′-CTATCTAACG₁TTCTCTGT-3′ 225′-CTATCTGAmCGTTCTCTGT-3′ 9 5′-CTATCTGUCGTTCTCTGT-3′ 105′-CTATCTGUCGTTCTCTGT-3′ 19 5′-CTATCTGUCG₁TTCTCTGT-3′ 385′-CTATCTGUCG₄TTCTCTGT-3′ 40 5′-CTATCTGUC₄GTTCTCTGT-3′ 425′-CTATCTGUC₅GTTCTCTGT-3′ 44 5′-CTATCTGUC₆GTTCTCTGT-3′ 465′-CTATCTGUCG₅TTCTCTGT-3′ 48 5′-CTATCTGUC₇GTTCTCTGT-3′ 665′-CTATCTAUCGTTCTCTGT-3′ 69 5′-CTATCTAUCG₁TTCTCTGT-3′ 655′-CTATCTAGCGTTCTCTGT-3′ 68 5′-CTATCTAGCG₁TTCTCTGT-3′ 235′-CTATCTGmACGTTCTCTGT-3′ 24 5′-CTATCTGmAmCGTTCTCTGT-3′ 255′-CTATCTGAC₂GTTCTCTGT-3′ 27 5′-CTATCTGTC₂GTTCTCTGT-3′ 335′-CTATCTGAC₃GTTCTCTGT-3′ 35 5′-CTATCTGTC₃GTTCTCTGT-3′ 265′-CTATCTGACG₂TTCTCTGT-3′ 28 5′-CTATCTGTCG₂TTCTCTGT-3′ 345′-CTATCTGACG₃TTCTCTGT-3′ 36 5′-CTATCTGTCG₃TTCTCTGT-3′ 495′-CTATCTAGCGTTCTCTGT-3′ 50 5′-CTATCTAGCGTTCTCTGT-3′ 65′-CTATCTGACGUUCTCTGT-3′ 51 5′-CTATCTAGCGTTCTCTGT-3′ 21 and3′-TCTTGCAGTCT-X₂-TCTGACGTTCT-3′ 21 52 5′-CCTACTAGCGTX₁CTCATC-3′ 535′-CCTACTAGCGX₁TCTCATC-3′ 54 5′-CCTACTAG₃CGTTCTCATC-3′ 555′-TCCATGA₁CGTTCCTGATGC-3′ 56 5′-CTATCTGAC₂G₂TTCTCTGT-3′ 575′-C₂T₂A₂T₂C₂T₂G₂A₂C₂G₂T₂T₂C₂T₂C₂T₂G₂T₂-3′ 29 5′-CTATCTGAX₁GTTCTCTGT-3′30 5′-CTATCTGACX₁TTCTCTGT-3′ 31 5′-CTATCTGTX₁GTTCTCTGT-3′ 325′-CTATCTGTCX₁TTCTCTGT-3′ 61 5′-CTATCTAGCGTX₁CTCTGT-3′ 625′-CTATCTAGCGX₁TCTCTGT-3′ 63 5′-CTATCTAGCGX₁X₁CTCTGT-3′ 585′-CTATCTGACGTX₃CTCTGT-3′ 59 5′-CTATCTGACGX₃TCTCTGT-3′ 605′-CTATCTGACGX₃X₃CTCTGT-3′ 70 5′-CTATCTAGCGTX₃CTCTGT-3′ 715′-CTATCTAGCGX₃TCTCTGT-3′ 72 5′-CTATCTAGCGX₃X₃CTCTGT-3′ 745′-CTATCTGACGTTCTCTGT-3′ 75 5′-CTATCTGACG₁ UUCTCTGT-3′ 765′-CCTACTAG₆CGTTCTCATC-3′ 77 5′-TCCATGACGU₁TCCTGATGC-3′ 785′-CTATCTGX₂CGTTCTCTGT-3′ 79 5′-CTATCTX₂ACGTTCTCTGT-3′ 805′-CTATCTU₂ACGTTCTCTGT-3′ 81 5′-CTATCTGU₂CGTTCTCTGT-3′ 825′-CTATCTGACGX₂TCTCTGT-3′ 83 5′-CTATCTGACGTX₂CTCTGT-3′ 845′-CTATCTGX₃CGTTCTCTGT-3′ 85 5′-CTATCTX3ACGTTCTCTGT-3′ 865′-(TCTGACGTTCT)₂X₂ 87 5′-(TCTGACG₁TTCT)₂X₂ 88 5′-(TCTGACG₄TTCT)₂X₂ 895′-(TCTCTGACGTT)₂X₂ 90 and 5′-TCTGACG₁TTCT-X₃-TGACCGGTCA-3′ 8 915′-CTATCTGTCGUUCTCTGT-3′ 92 5′-CTATCTGTCG₁ UUCTCTGT-3′ 935′-(TCTGUCGTTCT)₂X₂ 94 5′-(TCTGUCG₁TTCT)₂X₂ 95 5′-(TCTGACG₄TTCT)₂X₂ 965′-(TCTGACG₁TT)₂X₂ 97 and 5′-TCTGACG₁TTCT-X₃-TCAACCACACA-3′ 11 985′-CTATCTGACG₁TTCTCUGU-3′ 99 5′-CTATCTGUCG₁TTCTCUGU-3′ 1005′-(UGUCG₁TTCT)₂X₂ 101 5′-(UGACG₁TTCT)₂X₂ Underlined G, A or U = 2′-OMe;Underlined T = 3′-OMe; A₁ = 3′-OMe; G₁ = 7-deaza-dG; m = P-Me; A₂, T₂,C₂, and G₂ = B-L-deoxy nucleoside; X₁ = abasic; X₂ = glycerol linker, X₃= C3-linker; C₃ and G₃ = 3′-deoxy-nucleoside; G₄ = araG; C₄ = araC; C₅ =5-OH-dC; C₆ = 1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine; G₅ =N1-Me-dG; C₇ = N3-Me-dC; U₁ = 3′-OMe; U₂ = dU; IROs in which two copiesof a nucleotide sequence are linked by a linker X are indicated usingparentheses, e.g., 5′-(TCTGACGTTCT)₂X₂represents an IRO in which twocopies of the nucleotide sequence 5′-TCTGACGTTCT-3′ (SEQ ID NO: 86) arelinked by a linker X₂

In some embodiments, the oligonucleotides each have from about 6 toabout 35 nucleoside residues, preferably from about 9 to about 30nucleoside residues, more preferably from about 11 to about 23nucleoside residues. In some embodiments, the oligonucleotides have fromabout 6 to about 18.

In a second aspect, the invention provides pharmaceutical formulationscomprising an IRO compound according to the invention and aphysiologically acceptable carrier.

In a third aspect, the invention provides methods for inhibiting orsuppressing TLR-mediated induction of an immune response in avertebrate, such methods comprising administering to the vertebrate aIRO compound according to the invention. In some embodiments, thevertebrate is a mammal. In preferred embodiments, IRO compound isadministered to a vertebrate in need of immune suppression.

According to this aspect of the invention, an IRO compound is capable ofsuppressing a TLR-based immune response to a further TLR ligand or TLRagonist. As discussed further in the Examples below, the activation of aTLR-based immune response by a TLR agonist or TLR ligand (e.g. an immunemodulatory oligonucleotide) can be suppressed/inhibited by thesimultaneous, pre- or post-administration of an IRO compound, and suchsuppression/inhibition may be maintained for an extended period of time(e.g. days) after administration. This beneficial property of thecurrent invention has a unique advantage for the prevention and/ortreatment of a disease or disorder. For example, application of certainTLR-agonists in the course of treating the disease may cause unwantedimmune stimulation that an IRO compound could suppress/inhibit.Administration of the IRO simultaneously, pre and/or post administrationof the TLR-agonist may allow therapeutic benefits from the TLR-agonistwhile suppressing/inhibiting the unwanted side effect(s). Additionally,pre-administration of an IRO could prevent an immune response (e.g.,allergic reaction) to a subsequent or later challenge by a TLR-agonist.

In the methods according to this aspect of the invention, administrationof IRO compound can be by any suitable route, including, withoutlimitation, parenteral, mucosal delivery, oral, sublingual, transdermal,topical, inhalation, intranasal, aerosol, intraocular, intratracheal,intrarectal, vaginal, by gene gun, dermal patch or in eye drop ormouthwash form. Administration of the therapeutic compositions of IROcompound can be carried out using known procedures at dosages and forperiods of time effective to reduce symptoms or surrogate markers of thedisease. When administered systemically, the therapeutic composition ispreferably administered at a sufficient dosage to attain a blood levelof IRO compound from about 0.0001 micromolar to about 10 micromolar. Forlocalized administration, much lower concentrations than this may beeffective, and much higher concentrations may be tolerated. Preferably,a total dosage of IRO compound ranges from about 0.001 mg per patientper day to about 200 mg per kg body weight per day. It may be desirableto administer simultaneously, or sequentially a therapeuticallyeffective amount of one or more of the therapeutic compositions of theinvention to an individual as a single treatment episode.

The IRO compound may optionally be linked to one or more allergensand/or antigens (self or foreign), an immunogenic protein, such askeyhole limpet hemocyanin (KLH), cholera toxin B subunit, or any otherimmunogenic carrier protein. IRO can also be used in combination withother compounds (e.g. adjuvants) including, without limitation, TLRagonists (e.g. TLR2 agonists and TLR9 agonists), Freund's incompleteadjuvant, KLH, monophosphoryl lipid A (MPL), alum, and saponins,including QS-21 and imiquimod, or combinations thereof.

The methods according to this aspect of the invention are useful formodel studies of the immune system. The methods are also useful for theprophylactic or therapeutic treatment of human or animal disease. Forexample, the methods are useful for pediatric and veterinary vaccineapplications.

In a fourth aspect, the invention provides methods for therapeuticallytreating a patient having a disease or disorder, such methods comprisingadministering to the patient a IRO compound according to the invention.In various embodiments, the disease or disorder to be treated is cancer,an autoimmune disorder, infectious disease, airway inflammation,inflammatory disorders, allergy, asthma, or a disease caused by apathogen. Pathogens include bacteria, parasites, fungi, viruses,viroids, and prions. Administration is carried out as described for thethird aspect of the invention.

In a fifth aspect, the invention provides methods for preventing adisease or disorder, such methods comprising administering to thepatient IRO compound according to the invention. In various embodiments,the disease or disorder to be prevented is cancer, an autoimmunedisorder, airway inflammation, inflammatory disorders, infectiousdisease, allergy, asthma, or a disease caused by a pathogen. Pathogensinclude bacteria, parasites, fungi, viruses, viroids, and prions.Administration is carried out as described for the third aspect of theinvention.

In any of the methods according to this aspect of the invention, the IROcompound can be administered in combination with any other agent usefulfor treating the disease or condition that does not diminish the immunemodulatory effect of the IRO compound. In any of the methods accordingto the invention, the agent useful for treating the disease or conditionincludes, but is not limited to, one or more vaccines, antigens,antibodies, cytotoxic agents, allergens, antibiotics, antisenseoligonucleotides, TLR agonist, TLR antagonist, peptides, proteins, genetherapy vectors, DNA vaccines and/or adjuvants to enhance thespecificity or magnitude of the immune response, or co-stimulatorymolecules such as cytokines, chemokines, protein ligands,trans-activating factors, peptides and peptides comprising modifiedamino acids. For example, in the treatment of cancer, it is contemplatedthat the IRO compound may be administered in combination with one ormore chemotherapeutic compound, targeted therapeutic agent and/ormonoclonal antibody. Alternatively, the agent can include DNA vectorsencoding for antigen or allergen. In these embodiments, the IROcompounds of the invention can variously act as adjuvants and/or producedirect immune modulatory effects.

The following examples are intended to further illustrate certainexemplary embodiments of the invention and are not intended to limit thescope of the invention. For example, representative TLR-ligands areshown in the following examples, but do not limit the scope of ligandsto which the IROs of the invention act as antagonists.

EXAMPLE 1 Synthesis of Oligonucleotides Containing Immune RegulatoryMoieties

All IRO were synthesized according to standard procedures (see e.g. U.S.Patent Publication No. 20040097719).

Oligonucleotides were synthesized on a 1 μM scale using an automated DNAsynthesizer (Expedite 8909; PerSeptive Biosystems, Framingham, Mass.),following standard linear synthesis or parallel synthesis procedures(see e.g. FIGS. 5 and 6 of U.S. Patent Publication No. 20040097719).

Deoxyribonucleoside phosphoramidites were obtained from (Aldrich-Sigma,St Louis, Mo.). 1′,2′-dideoxyribose phosphoramidite,propyl-1-phosphoramidite, 2-deoxyuridine phosphoramidite,1,3-bis-[5-(4,4′-dimethoxytrityl)pentylamidyl]-2-propanolphosphoramidite and methyl phosponamidite were obtained from GlenResearch (Sterling, Va.). .beta.-L-2′-deoxyribonucleosidephosphoramidite, .alpha.-2′-deoxyribonucleoside phosphoramidite,mono-DMT-glycerol phosphoramidite and di-DMT-glycerol phosphoramiditewere obtained from ChemGenes (Willmington, Mass.).(4-Aminobutyl)-1,3-propanediol phosphoramidite was obtained fromClontech (Palo Alto, Calif.). Arabinocytidine phosphoramidite,arabinoguanosine, arabinothymidine and arabinouridine were obtained fromReliable Pharmaceutical (St. Louis, Mo.). Arabinoguanosinephosphoramidite, arabinothymidine phosphoramidite and arabinouridinephosphoramidite were synthesized at Idera Pharmaceuticals, Inc.(Cambridge, Mass.) (Noronha et al. (2000) Biochem., 39:7050-7062).

All nucleoside phosphoramidites were characterized by ³¹P and ¹H NMRspectra. Modified nucleosides were incorporated at specific sites usingnormal coupling cycles. After synthesis, oligonucleotides weredeprotected using concentrated ammonium hydroxide and purified byreverse phase HPLC, followed by dialysis. Purified oligonucleotides assodium salt form were lyophilized prior to use. Purity was tested by CGEand MALDI-TOF MS.

EXAMPLE 2 Inhibition of TLR9 Stimulation

HEK293 cells stably expressing TLR9 (Invivogen) were transientlytransfected with reporter gene, Seap, (Invivogen) for 6 hr. Cells weretreated with 0.5 μg/ml 5′-CTATCTGACGTTCTCTGT-3′ (mouse CpG sequence;IMO/SEQ ID NO 1; 0 dose) alone and various concentrations of IRO 5 or 6for 18 hr. TLR9-dependent reporter gene expression was determinedaccording to the manufacturer's protocol (Invivogen) and the results areexpressed as % activity of TLR9 stimulating oligonucleotide (100%). Theresults are shown in FIG. 1. These results demonstrate that IRO 5inhibited TLR9 agonistic activity of IMO.

EXAMPLE 3 IRO Specifically Inhibit TLR9 Stimulation

HEK293 cells stably expressing TLR9 or TLR3 (Invivogen) were transientlytransfected with reporter gene, Seap, (Invivogen) for 6 hr. Cells weretreated with 0.5 mg/ml IMO1 (0.5 μg/ml), IRO 5 (2.0 μg/ml), R848 (5.0μg/ml), or poly (I).poly(C) (0.5 μg/ml) and combinations of IMO+IRO,R848+IRO, or poly(I).poly(C)+IRO for 18 hr. TLR9- or TLR3-dependentreporter gene expression was determined according to the manufacturer'sprotocol (Invivogen) and the results are expressed as fold change inNF-kB activity. The results are shown in FIG. 2. These resultsdemonstrate that IRO 5 inhibits the activity of the TLR9 agonist but notagonist of TLR3, and more generally that IRO's can selectively inhibitTLR activation.

EXAMPLE 4 Dose-dependent Inhibition by IRO

C57BL/6 mice were injected subcutaneously (s.c.) at left underarm with0.25 mg/kg stimulating 5′-TCTGACG₁TTCT-X-TCTTG₁CAGTCT-5′ (IMO/SEQ ID NO3; G₁=7-deazaG, X=glycerol) and different doses of IRO 5 at right underarm. Serum samples were taken at 2 hours after stimulating IMO3injection and determined IL-12 levels by ELISA. The results are shown inFIG. 3. These results demonstrate dose-dependent inhibition by IRO.

EXAMPLE 5 Time-dependence Inhibition by IRO

C57BL/6 mice were injected s.c. at left underarm with 0.25 mg/kgstimulating IMO 3 and 1 mg/kg IRO 5 or 5′-CTATCTCACCTTCTCTGT-5′ (non-CpGnon-stimulatory control; oligo/SEQ ID NO 4) at right under arm eitherone hour before (−1 h) or at the same time as stimulating IMO (0 h).Serum samples were taken at 2 hours after stimulating IMO injection anddetermined IL-12 levels by ELISA. The results in FIG. 4A demonstrate adecrease in serum IL-12 levels after administration of IRO 5 or (oligo4) either one hour before (−1 h) or at the same time as stimulating IMO(0 h).

C57BL/6 mice were injected s.c. at left underarm with 0.25 mg/kgstimulating IMO 3 and intranasal administration of 10 mg/kg IRO 102 atthe same time as stimulating IMO (0 h). Serum samples were taken at 2hours after stimulating IMO injection and determined IL-12 levels byELISA. The results in FIG. 4B demonstrate a decrease in serum IL-12levels after intranasal administration of IRO 102 at the same time ass.c. of IMO.

C57BL/6 mice were injected s.c at left underarm with 0.25 mg/kgstimulating IMO 3 and 2 mg/kg or 10 mg/kg IRO 17, 99, 102 s.c. at rightunder arm either one hour before (−1 h), twenty-four hours before (−24)or seventy-two hours before (−72) as stimulating IMO (0 h). Serumsamples were taken at 2 hours after stimulating IMO injection anddetermined IL-12 levels by ELISA. The results are shown in FIG. 4C-D.These results demonstrate pre-administration and simultaneousadministration of IRO was able to inhibit agonist of TLR9, and moregenerally that IRO's can inhibit TLR activation.

EXAMPLE 6 Inhibition of TLR9 Stimulation

C57BL/6 mice were injected s.c. at left underarm with 0.25 mg/kgstimulating IMO 3 and 1 mg/kg IRO 21 or control oligo 4 at right underarm either one hour before (−1 h) or at the same time as stimulating IMO(0 h). Serum samples were taken at 2 hours after stimulating IMOinjection and determined IL-12 levels by ELISA. The results are shown inFIGS. 5A and 5B. These results demonstrate that a CpG oligonucleotidelinked at its 5′ ends show inhibitory properties, and more generallythat immune stimulatory CpG oligonucleotides linked at their 5′ ends caninhibit TLR activation.

EXAMPLE 7 Inhibition of TLR9 in Human Cell Cultures

Human pDCs and PBMCs were incubated with 10 ug 5′-CTATCTGTCGTTCTCTGT-3′(human CpG sequence; IMO/SEQ ID NO 2) and 40 ug IRO 10 for 24 hr. Theresults are shown in FIG. 6. These results demonstrate that an IROinhibited TLR9 agonist activity in human cell cultures, and moregenerally that IROs can inhibit TLRs in human cells.

EXAMPLE 8 IRO Effect on OVA Induced Th2 Immune Response

The results are shown in FIG. 7. These results demonstrate that an IROdoes not have an effect on Ovalbumin (“OVA”) induced Th2 immuneresponses, whereas IMO compounds reduce OVA induced Th2 response andcause the production of Th1 cytokines.

EXAMPLE 9 IRO Inhibition of IMO Effects on Th1 and Th2 Immune Responses

The results are shown in FIG. 8. These results demonstrate that an IROcan reverse Th2 inhibitory properties and can inhibit Th1 immuneresponses induced by IMO.

EXAMPLE 10 Antibody Responses to IMO and IRO

Mice were immunized with HBsAg in the presence and absence of IMO 1 andIRO 5 or 6 and combinations thereof at wk 0 and wk 2 and antibodyresponses were measured wk 4. The results are shown in FIG. 9 anddemonstrate reduction by an IRO on an IMO induced IgG2A immune response.

EXAMPLE 11 Inhibition of Immune Stimulatory Oligonucleotides

HEK293 cells stably expressing TLR9 (Invivogen) were transientlytransfected with reporter gene, Seap, (Invivogen) for 6 hr. Cells weretreated with 0.25 μg/ml IMO alone (IMO 1; 0 dose) and variousconcentrations of IROs for 18 hr. TLR9-dependent reporter geneexpression was determined according to the manufacturer's protocol(Invivogen) and the results are expressed as % inhibition of immunestimulating oligonucleotide activity. The results are shown in Tables 5and 6 below. These results demonstrate that IROs inhibited activity ofIMO.

TABLE 5 Percent inhibition of immune stimulatory oligo-nucleotide 1. IIMO1 concentration was 0.25μg/ml and IRO concentration was 2 μg/ml SEQ ID NO:/ IRO # Sequence% Inhibition 5 5′-CTATCTGACGTTCTCTGT-3′ 52.5% 255′-CTATCTGAC₂GTTCTCTGT-3′ 17.5% 26 5′-CTATCTGACG₂TTCTCTGT-3′ 15.3% 335′-CTATCTGAC₃GTTCTCTGT-3′ 38.1% 39 5′-CTATCTGAC₄GTTCTCTGT-3′ 52.8% 415′-CTATCTGAC₅GTTCTCTGT-3′ 42.6% 43 5′-CTATCTGAC₆GTTCTCTGT-3′ 23.6% IROscontaining various modifications inhibit NF-κB activation of IMO inHEK293 cells expressing TLR9, and more generally IROs containing variousmodifications can inhibit NF-κB activation of IMO.

TABLE 6 Percent inhibition of immune stimulatory oligo-nucleotide 1. IMO1 concentration was 0.25 μg/mland IRO concentration was 3 μg/ml. SEQ ID NO:/ IRO # Sequence% Inhibition 5 5′-CTATCTGACGTTCTCTGT-3′ 76.5% 175′-CTATCTGACG₁TTCTCTGT-3′ 76.4% 34 5′-CTATCTGACG₃TTCTCTGT-3′ 32.2% 37.5′-CTATCTGACG₄TTCTCTGT-3′ 78.3% IROs containing various modificationsinhibit NF-κB activation of IMO in HEK293 cells expressing TLR9, andmore generally IROs containing various modifications can inhibit NF-κBactivation of IMO.

EXAMPLE 12 Time-dependence Inhibition by IRO

C57BL/6 mice were injected subcutaneously (s.c.) at left underarm with0.25 mg/kg to 10 mg/kg TLR agonist and 1 mg/kg to 20 mg/kg IRO 5, 17 or37 or 5′-TCCTGGCGGGGAAGT-3′ (poly dG control; oligo/SEQ ID NO 12) atright under arm at one hour (−1 h) or up to forty-eight hours (−48)before or at the same time as TLR agonist (0 h). Serum samples weretaken at 2 hours after stimulating IMO injection and determined IL-12levels by ELISA. The results are shown in Tables 7-22 below. Theseresults demonstrate that both pre-administration and simultaneousadministration of an IRO inhibits agonists of TLR9, and that theinhibitory activities of an IRO were effective even when administered 48hours prior to the administration of the IMO. More generally, theseresults demonstrate that pre-administration and simultaneousadministration of an IRO can both inhibit TLR agonists and that theinhibitory activities of an IRO can be seen even when administered manyhours prior to the administration of the TLR agonist.

TABLE 7 Inhibition of IMO 3 induced IL-12 (ng/ml ± SD) by IRO 5 in vivo,C57BL/6 mice (n = 3) IMO IRO IRO + IMO alone alone Time of IMOadministration after IRO administration (0.25 mg/kg) (2 mg/kg) 0 hr 1 hr3 hr 6 hr 21.1 ± 1.84 0.81 ± 0 0.59 ± 0.48 1.54 ± 0.17 6.53 ± 0.81 10.41± 0.48 IRO 5 inhibited IMO induced IL-12 production when injected up to6 hr after IRO administration. More generally, these results demonstratethat an IRO can inhibit TLR activation and IMO induced IL-12 productionwhen IMO is administered or initially becomes present hours after IROadministration.

TABLE 8 Inhibition of IMO 3 induced IL-12 (ng/ml ± SD) by IRO 5 in vivo,C57BL/6 mice (n = 3) IMO IRO IRO + IMO alone alone Time of IMOadministration after IRO administration (0.25 mg/kg) (20 mg/kg) 0 hr 1hr 3 hr 6 hr 33.8 ± 3.8 0.73 ± 0.7 0.87 ± 1.19 1.52 ± 2.01 2.2 ± 2.41.84 ± 3.18 IRO 5 potently inhibited IMO induced IL-12 production wheninjected up to 6 hr after IRO administration. More generally, theseresults demonstrate that an IRO can substantially inhibit TLR activationand IMO induced IL-12 production when IMO is administered or initiallybecomes present hours after IRO administration.

TABLE 9 Inhibition of IMO 3 induced IL-12 (ng/ml ± SD) by IRO 5 in vivo,C57BL/6 mice (n = 3) IRO IRO + IMO IMO alone Time of IMO administrationalone (20 mg/ after IRO administration (0.25 mg/kg) kg) 6 hr 14 hr 24 hr48 hr 25.8 ± 2.6 0.17 ± 0 0.04 ± 0 1.25 ± 0 1.8 ± 0.29 2.9 ± 0.1 IRO 5potently inhibited IMO induced IL-12 production when injected up to 48hr after IRO administration. More generally, these results demonstratethat an IRO can substantially inhibit TLR activation and IMO inducedIL-12 production when IMO is administered or initially becomes presenthours after IRO administration.

TABLE 10 Inhibition of IMO 3 induced IL-12 (ng/ml ± SD) by IRO 17 invivo, C57BL/6 mice (n = 3) RO + IMO IMO IRO Time of IMO administrationalone alone after IRO administration (0.25 mg/kg) (2 mg/kg) 3 hr 6 hr 24hr 6.6 ± 0.64 0.67 ± 0.02 1.01 ± 0.06 1.25 ± 0.29 4.29 ± 1.12 IRO 17inhibited IMO induced IL-12 production when injected up to 6 hr or moreafter IRO administration. More generally, these results demonstrate thatan IRO can inhibit TLR activation and IMO induced IL-12 production whenIMO is administered or initially becomes present hours after IROadministration.

TABLE 11 Inhibition of IMO 3 induced IL-12 (ng/ml ± SD) by IRO 37 invivo, C57BL/6 mice (n = 3) IRO + IMO IMO IRO Time of IMO administrationalone alone after IRO administration (0.25 mg/kg) (2 mg/kg) 3 hr 6.6 ±0.64 0.67 ± 0.02 0.91 ± 0.03 IRO 37 inhibited IMO induced IL-12production when injected up to 3 hr after IRO administration. Moregenerally, these results demonstrate that an IRO can inhibit TLRactivation and IMO induced IL-12 production when IMO is administered orinitially becomes present hours after IRO administration.

TABLE 12 Inhibition of IMO 3 induced IL-12 (ng/ml ± SD) by control polydG (5′-TCCTGGAGGGGAAGT-3′ (SEQ ID NO 73)) in vivo, C57BL/6 mice (n = 3)Control + IMO IMO IRO Time of IMO administration alone alone afterControl administration (0.25 mg/kg) (10 mg/kg) 3 hr 6 hr 24 hr 18.24 ±0.22 1.47 ± 0 1.38 ± 0.18 10.03 ± 0.37 16.97 ± 0.52 A poly dG compoundknown to show TLR9 antagonist activity inhibited IMO induced IL-12production when injected up to 6 hr after IRO administration. Comparedwith the data for IRO (e.g. IRO 5 in Table 7), control poly dG oligoantagonistic effects are short-term and transient.

TABLE 13 Inhibition of IMO 3 induced IL-12 (ng/ml ± SD) by control polydG (5′-TCCTGGCGGGGAAGT-3′ (SEQ ID NO 12)) in vivo, C57BL/6 mice (n = 3)Control + IMO IMO IRO Time of IMO administration alone alone afterControl administration (0.25 mg/kg) (10 mg/kg) 3 hr 6 hr 24 hr 18.24 ±0.22 1.2 ± 0 0.81 ± 0.06 10.1 ± 0.09 19.02 ± 1.6 A poly dG compoundknown to show TLR9 antagonist activity inhibited IMO induced IL-12production when injected up to 6 hr after IRO administration. Comparedwith the data for IRO (e.g. IRO 5 in Table 7), control poly dG oligoantagonistic effects are short and transient.

TABLE 14 Inhibition of R848, a TLR7 and TLR8 agonist, induced IL-12(ng/ml ± SD) by IRO 5 in vivo, C57BL/6 mice (n = 3) IRO + R848 R848 IROTime of R848 administration alone alone after IRO administration (0.5mg/kg) (2 mg/kg) 1 hr 128 ± 2.9 1.48 ± 0.17 56.0 ± 3.3 IRO 5 shows a lowtransient inhibition of R848 induced IL-12 production when injected upto 1 hr after IRO administration. More generally, these data demonstratethat an IRO can inhibit activity of intracellular TLRs.

TABLE 15 Inhibition of PolyI:PolyC, a TLR3 agonist, induced IL-12 (ng/ml± SD) by IRO 5 in vivo, C57BL/6 mice (n = 3) PolyI.PolyC IRO +PolyI.PolyC alone IRO Time of PolyI,.PolyC administration aloneadministration after IRO (10 mg/kg) (2 mg/kg) 1 hr 8.7 ± 0.6 1.48 ± 0.172.1 ± 0.07 IRO 5 shows a low transient inhibition of PolyI.PolyC inducedIL-12 production when injected up to 1 hr after IRO administration. Moregenerally, these data demonstrate that an IRO can inhibit TLR activationand PolyI.PolyC induced IL-12 production.

TABLE 16 Inhibition of IMO induced MCP-1 (ng/ml ± SD) by IRO 5 in vivo,C57BL/6 mice (n = 3) IRO + IMO IMO IRO Time of R848 administration alonealone after IRO administration (0.25 mg/kg) (2 mg/kg) 1 hr 2.2 ± 0.25 NT0.28 ± 0.73 IRO 5 shows potent inhibition of IMO induced MCP-1production when injected up to 1 hr after IRO administration. Moregenerally, these data demonstrate that an IRO can inhibit TLR activationand IMO induced MCP-1 production.

TABLE 17 Inhibition of R848, a TLR7 and TLR8 agonist, induced MCP-1(ng/ml ± SD) by IRO 5 in vivo, C57BL/6 mice (n = 3) IRO + R848 R848 IROTime of R848 administration alone alone after IRO administration (0.5mg/kg) (2 mg/kg) 1 hr 11 ± 1.4 7.2 ± 1.7 IRO 5 shows a low transientinhibition of R848 induced MCP-1 production when injected up to 1 hrafter IRO administration. More generally, these data demonstrate that anIRO can inhibit TLR activation and MCP-1 production throughintracellular TLRs.

TABLE 18 Inhibition of PolyI.PolyC, a TLR3 agonist, induced MCP-1 (ng/ml± SD) by IRO 5 in vivo, C57BL/6 mice (n = 3) IRO + PolyI.PolyCPolyI.PolyC IRO Time of PolyI.PolyC administration alone alone after IROadministration (10 mg/kg) (2 mg/kg) 1 hr 4.6 ± 0.6 1.8.0 ± 0.57 IRO 5shows a low transient inhibition of PolyI.PolyC induced MCP-1 productionwhen injected up to 1 hr after IRO administration. More generally, thesedata demonstrate that an IRO can inhibit TLR activation and MCP-1production of a PolyI.PolyC

TABLE 19 Inhibition of IMO 3 induced IL-12 (ng/ml ± SD) by IRO 5 invivo, C57BL/6 mice (n = 3) IRO + IMO IMO IRO Time of IMO administrationalone alone after IRO administration (0.25 mg/kg) (20 mg/kg) 2 days 5days 7 days 33.2 ± 8.7 NT 14.5 ± 5.17 17.19 ± 11.2 28.0 ± 7.75 IRO 5shows potent inhibition of IMO induced IL-12 production when injected upto 7 days after IRO administration. More generally, these datademonstrate that an IRO can inhibit TLR activation and IMO induced IL-12production in mammals.

TABLE 20 Inhibition of IMO induced IL-12 (ng/ml ± SD) by IRO 5 in vivo,C57BL/6 mice (n = 3) IRO + IMO IMO IRO Time of IMO administration alonealone after IRO administration (0.25 mg/kg) (10 mg/kg) 72 hr 53.39 ±2.71 2.03 ± 2.03 28.72 ± 0.79 IRO 5 shows potent inhibition of IMOinduced IL-12 production when injected up to 72 hr after IROadministration. More generally, these data demonstrate that an IRO caninhibit TLR activation and IMO induced IL-12 production in mammals hoursafter the IRO is administered.

TABLE 21 Inhibition of R848, a TLR7 and TLR8 agonist, induced IL-12(ng/ml ± SD) by IRO 5 in vivo, C57BL/6 mice (n = 3) IRO + R848 R848 IROTime of R848 administration alone alone after IRO administration (0.125mg/kg) (10 mg/kg) 72 hr 96.5 ± 3.4 2.03 ± 2.03 13.64 ± 0.47 IRO 5 showsinhibition of R848 induced IL-12 production when injected up to 72 hrafter IRO administration. More generally, these data demonstrate that anIRO can inhibit the activity of an agonist of intracellular TLR's andTLR agonist induced IL-12 production in mammals hours after the IRO isadministered.

TABLE 22 Inhibition of PolyI.PolyC, a TLR3 agonist, induced IL-12 (ng/ml± SD) by IRO 5 in vivo, C57BL/6 mice (n = 3) IRO + PolyI.PolyCPolyI.PolyC IRO Time of PolyI.PolyC administration alone alone after IROadministration (10 mg/kg) (10 mg/kg) 72 hr 28.42 ± 1.2 2.03 ± 2.03 26.61± 5.97 IRO 5 shows no inhibition of PolyI.PolyC induced IL-12 productionwhen injected 72 hr after IRO administration.

EXAMPLE 13 Short-term and Long-term Blocking Activities of IRO AgainstTLR Agonist

To assess the short term activity and selectivity of IRO compounds, micewere subcutaneously injected with 2 mg/kg IRO in their right flank onehour (−1 h) before subcutaneous administration of a TLR agonist to theleft flank. Serum samples were taken at 2 hours after administration ofthe TLR agonist and were analyzed using multiple cytokine/chemokinedetecting Luminex kits obtained from Biosource (Camarillo, Calf.).Manufacture recommended protocols were followed. Cytokine/chemokinevalues were determined from mean values falling on the standard curvedetermined on a Luminex 100 instrument. Luminex analysis was performedusing STarStation software (Applied Cytometry Systems, Sacramento,Calf.). The following representative agonists were used at the indicateddose: 5′-TCTGACG₁TTCT-X-TCTTG₁CAGTCT-5′ (SEQ ID NO: 3) (TLR9 agonist;0.25 mg/kg, G₁=7-deaza-dG), R848 (TLR7/8 agonist, 0.1 mg/kg), Loxoribine(TLR7 agonist, 100 mg/kg), Flagellin (TLR5 agonist, 0.25 mg/kg), LPS(TLR4 agonist, 0.25 mg/kg), PolyI.PolyC (TLR3 agonist, 20 mg/kg), andMALP-2 (TLR2 agonist, 0.5 mg/kg). The results are shown in FIGS. 10-12.These data demonstrate that IROs can inhibit cytokine/chemokineproduction in response to TLR agonists. The effect is greater forintracellular TLRs (e.g. TLR3, TLR7, TLR8, and TLR9) as compared toextracellular TLRs (e.g. TLR2, TLR4, and TLR5).

To assess the long-term activity and selectivity of IRO compounds, micewere subcutaneously injected with 10 mg/kg IRO in their right flankseventy-two hours (−72 h) before subcutaneous administration of a TLRagonist (as described above) to the left flank. Serum samples were takenat 2 hours after administration of the TLR agonist and were analyzed asdescribed above. The results are shown in FIGS. 13-15. These resultsdemonstrate pre-administration administration of an IRO was able toinhibit TLR agonist, and that the inhibitory activities of IRO wereeffective even when administered 72 hours prior to the administration ofthe agonist.

EXAMPLE 14 Activities of IRO Compounds in Lupus Mouse Model

Purified mouse spleen B cells from wild-type (BALB/c) and lupus prone(MRL-lpr) mice were cultured with 1 μg/ml IRO-17 in the presence orabsence of 0.3 μg/ml IMO, or 0.3 μg/ml IMO or medium alone for 72 h. Theresults are shown in FIG. 16. These results demonstrate thatadministration of IRO was able to inhibit B lymphocyte proliferation.

Purified mouse spleen B cells from wild-type (BALB/c) and lupus prone(MRL-lpr) mice were cultured with 1 μg/ml IRO-17 in the presence orabsence of 0.3 μg/ml IMO, or 0.3 μg/ml IMO or medium alone for 72 h. Theresults are shown in FIG. 17A. These results demonstrate thatadministration of IRO was able to inhibit IL-6 production by mice Blymphocytes. Purified mouse spleen B cells from wild-type (BALB/c) andlupus prone (NZBW) mice were cultured with 0.01 to 10 μg/ml IRO-17 inthe presence of 1 μg/ml IMO, or alone with 10 μg/ml IRO-17, 1 μg/ml IMOor medium for 72 h. The results are shown in FIGS. 17B and 17C. Theseresults demonstrate that administration of an IRO was able to inhibitIL-6 and IL-12 production by mice spleen cells.

Lupus prone MRL-lpr mice were injected once a week s.c. with 100 μgdoses of IRO-5 from wk 9 to 18, and 21 to 23 or IRO-17 starting from wk10 to 15, 100 μg three times week in weeks 18-21 and 40 mg three times aweek in weeks 22 to 24. Blood and urine were collected every week beforeIRO injection. Mice were sacrificed Wk 24. Serum anti-DNA IgG1 levelswere determined by ELISA. The results are shown in FIGS. 18A through18E. These results demonstrate that IRO 5 and IRO 17 can inhibit IgG1and IgG2A production and urine protein in Lupus prone mice.

Lupus prone NZBW mice are dosed with 300 μg IRO-5, s.c once in every twoweeks starting week 6. Serum anti-DNA IgG2a levels were determined atweeks 16 and 20. The results are shown in FIG. 19. These resultsdemonstrate that administration of IRO inhibits serum anti-DNA IgG2a inNZBW mice.

1. A method for reducing anti-DNA antibody levels in a vertebrate havinglupus, such method comprising administering to the vertebrate an immuneregulatory oligonucleotide (IRO) compound in a pharmaceuticallyeffective amount, wherein the IRO comprises at least twooligonucleotides linked by a non-nucleotide linker at their 3′ ends orby a functionalized sugar or by a functionalized nucleobase via anon-nucleotide linker, wherein at least one oligonucleotide has thestructure5′-N_(m)—N₃N₂N₁CGN¹N²N³—N^(m)-3′ wherein: CG is an oligonucleotide motifthat is CpG, C*pG, C*pG* or CpG*, wherein C is cytosine, C* is apyrimidine nucleotide derivative, G is guanosine, and G* is a purinenucleotide derivative; N₁ is a modified nucleotide that suppresses theactivity of the oligonucleotide motif selected from the group consistingof 2′-substituted ribonucleoside, 2′-O-substituted ribonucleoside,2′-substituted arabinoside, and 2′-O-substituted arabinoside; N₂—N₃and/or N¹—N³, at each occurrence, is independently i) a nucleotide, ii)a nucleotide derivative, or iii) a modified nucleotide that suppressesthe activity of the oligonucleotide motif selected from the groupconsisting of 2′-substituted ribonucleoside, 2′-O-substitutedribonucleoside, 2′-substituted arabinoside, and 2′-O-substitutedarabinoside; N_(m) and N^(m), at each occurrence, is independently anucleotide, nucleotide derivative or non-nucleotide linkage; andprovided that the compound contains less than 3 consecutive guanosinenucleotides; wherein the oligonucleotide motif would he immunestimulatory but for the one or more modified nucleotides that suppressesthe activity of the oligonucleotide motif; and wherein m is a numberfrom 0 to about 30 and wherein the IRO is an antagonist of an agonist ofTLR7, TLR8 and/or TLR9.
 2. A method for therapeutically treating avertebrate having lupus, multiple sclerosis, type I diabetes mellitus,irritable bowel syndrome, Chrohn's disease, arthritis, septic shock ormalaria, such method comprising administering to the vertebrate animmune regulatory oligonucleotide (IRO) compound in a pharmaceuticallyeffective amount, wherein the IRO comprises at least twooligonucleotides linked by a non-nucleotide linker at their 3′ ends orby a functionalized sugar or by a functionalized nucleobase, wherein atleast one oligonucleotide has the structure5′-N_(m)—N₃N₂N₁CGN¹N²N³—N^(m)-3′ wherein: CG is an oligonucleotide motifthat is CpG, C*pG, C*pG* or CpG*, wherein C is cytosine, C* is apyrimidine nucleotide derivative, G is guanosine, and G* is a purinenucleotide derivative; N₁ is a modified nucleotide that suppresses theactivity of the oligonucleotide motif selected from the group consistingof 2′-substituted ribonucleoside, 2′-O-substituted ribonucleoside,2′-substituted arabinoside, and 2′-O-substituted arabinoside; N₂—N₃and/or N¹—N³, at each occurrence, is independently i) a nucleotide, ii)a nucleotide derivative, or iii) a modified nucleotide that suppressesthe activity of the oligonucleotide motif selected from the groupconsisting of 2′-substituted ribonucleoside, 2′-O-substitutedribonucleoside, 2′-substituted arabinoside, and 2′-O-substitutedarabinoside; N_(m) and N^(m), at each occurrence, is independently anucleotide, nucleotide derivative or non-nucleotide linkage; andprovided that the compound contains less than 3 consecutive guanosinenucleotides; wherein the oligonucleotide motif would be immunestimulatory but for the one or more modified nucleotides that suppressesthe activity of the oligonucleotide motif; and wherein in is a numberfrom 0 to about 30 and wherein the IRO is an antagonist of an agonist ofTLR7, TLR8 and/or TLR9.
 3. The method according to claim 1 or 2, whereinthe IRO compound is administered in combination with one or morevaccines, antigens, antibodies, cytotoxic agents, allergens,antibiotics, antisense oligonucleotides, TLR agonists, TLR antagonists,peptides, proteins, gene therapy vectors, DNA vaccines, adjuvants orco-stimulatory molecules.
 4. The method according to claim 1 or 2,wherein the route of administration is parenteral, mucosal delivery,oral, sublingual, transdermal, topical, inhalation, intranasal, aerosol,intraocular, intratracheal, intrarectal, vaginal, by gene gun, dermalpatch or in eye drop or mouthwash form.
 5. The method according to claim1 or 2, wherein the non-nucleotide linker linking the at least twooligonucleotides at their 3′ ends or functionalized base or sugar isGlycerol (1,2,3-Propanetriol), 1,2,4, Butanetriol,2-(hydroxymethyl)-1,3-propanediol, 2-(hydroxymethyl)1,4-butanediol,1,3,5-Pentanetriol, 1,1,1-Tris(hydroxymethy)ethane,1,1,1-Tris(hydroxymethy)nitromethane, 1,1,1-Tris(hydroxymethyl)propane,1,2,6-Hexanetriol, 3-Methyl-1,3,5-pentanetriol, 1,2,3-Heptanetriol,2-Amino-2-(hydroxymethyl)-1,3-propanediol,N-[Tris(hydroxymethyl)methyl]acrylamide, cis-1,3,5-Cyclohexanetriol,Cis-1,3,5-Tri(hydroxymethyl)cyclohexane, 3,5-Di(hydroxymethyl)phenol,1,3,5-Trihydroxyl-benzene, 3,5-Di(hydroxymethy)benzene,1,3-Di(hydroxyethoxy)-2-hydroxyl-propane,1,3-Di(hydroxypropoxy)-2-hydroxyl-propane, 2-Deoxy-D-ribose,1,2,4-Trihydroxyl-benzene, D-Galactoal, 1,6-anhydro-β-D-Glucose,1,3,5-Tris(2-hydroxyethyl)-Cyanuric acid, Gallic acid,3,5,7-Trihydroxyflavone, 4,6-Nitropyrogallol, Ethylene glycol,1,3-Propanediol, 1,2-Propanediol, 1,4-Butanediol, 1,3-Butanediol,2,3-Butanediol, 1,4-Butanediol, 1,5-Pentanediol, 2,4-Pentanediol,1,6-Hexanediol, 1,2-Hexanediol, 1,5-Hexanediol, 2,5-Hexanediol,1,7-Heptanediol, 1,8-Octanediol, 1,2-Octanediol, 1,9-Nonanediol,1,12-Dodecanediol, Triethylene glycol, Tetraethylene glycol,2-(1-Aminopropyl)-1,3-propanediol, or 1,2-Dideoxyribose.
 6. The methodaccording to claim 1 or 2, wherein the non-nucleotide linker linking theat least two oligonucleotides at their 3′ ends or functionalized base orsugar is Glycerol (1,2,3-Propanetriol).
 7. The method according to claim1 or 2, wherein the pyrimidine nucleotide derivative is2′-deoxythymidine,1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine,2-dideoxy-5-halocytosine, 2′-dideoxy-5-nitrocytosine, arabinocytidine,2′-deoxy-5-hydroxycytidine, 2′-deoxy-N4-alkyl-cytidine,2′-deoxy-4-thiouridine, or other pyrimidine nucleoside analogs.
 8. Themethod according to claim 1 or 2, wherein the purine nucleotidederivative is 2′-deoxy-7-deazaguanosine, 2′-deoxy-6-thioguanosine,arahinoguanosine, 2′-deoxyinosine, or other purine nucleoside analogs.9. The method according to claim 1 or 2, wherein at least one of theoligonucleotides has the sequence TCTGACGTTCT (SEQ ID NO: 86),TCTGACG₁TTCT (SEQ ID NO: 87), TCTGACG₄TTCT (SEQ ID NO: 88), TCTCTGACGTT(SEQ ID NO: 89), TCTGUCGTTCT (SEQ ID NO: 93), TCTGUCG₁TTCT (SEQ ID NO:94), TCTGACG₄TTCT (SEQ ID NO: 95), TCTGACG₁TT (SEQ ID NO: 96),UGUCG₁TTCT (SEQ ID NO: 100), and UGACG₁ITTCT (SEQ ID NO: 101), whereinG₁=7-deaza, G₄=araG and G, A or U=2′-OMe.
 10. The method according toclaim 1 or 2, wherein the IRO is selected from5′-(TCTGACGTTCT)₂X₂(5′-SEQ ID NO: 86-3′-X₂-3′-SEQ ID NO: 86-5′),5′-(TCTGACG₁TTCT)₂X₂(5′-SEQ ID NO: 87-3′-X₂-3′-SEQ ID NO: 87-5′),5′-(TCTGACG₄TTCT)₂X₂(5′-SEQ ID NO: 88-3′-X₂-3′-SEQ ID NO: 88-5′),5′-(TCTCTGACGTT)₂X₂(5′-SEQ ID NO: 89-3′-X₂-3′-SEQ ID NO: 89-5′),5′-(TCTGUCGTTCT)₂X₂(5′-SEQ ID NO: 93-3′-X₂-3′-SEQ ID NO: 93-5′),5′-(TCTGUCG₁TTCT)₂X₂(5′-SEQ ID NO: 94-3′-X₂-3′-SEQ ID NO: 94-5′),5′-(TCTGACG₄TTCT)₂X₂(5′-SEQ ID NO: 95-3′-X₂-3′-SEQ ID NO: 95-5′),5′-(TCTGACG₁TT)₂X₂(5′-SEQ ID NO: 96-3′-X₂-3′-SEQ ID NO: 96-5′),5′-(UGUCG₁TTCT)₂X₂(5′-SEQ ID NO: 100-3′-X₂-3′-SEQ ID NO: 100-5′) and5′-(UGACG₁TTCT)₂X₂(5′-SEQ ID NO: 101-3′-X₂-3′-SEQ ID NO: 101-5′),wherein G₁=7-deaza, G₄=araG, G, A or U=2′-OMe and X₂=glycerol linker.11. The method according to claim 1 or 2, wherein the 2′-O-substitutedribonucleoside is a 2′-OMe-ribonucleoside.
 12. The method according toclaim 1 or 2, wherein the non-nucleotide linker may he attached to the3′ hydroxyl.
 13. The method according to claim 1 or 2, wherein CG is anoligonucleotide motif that is C*pG, C*pG* or CpG*.